The invention relates to a process for the detection, identification and quantification of hydrophobic proteins, protein fragments and modifications, and of hydrophobic peptides.
The quantification of hydrophobic proteins is not possible or is only inadequately possible using conventionally customary techniques. The customary methods for separation of hydrophilic proteins, such as Western blotting, are often only applicable to hydrophobic proteins to a limited extent, because on the one hand separation on SDS gel is inadequate and on the other hand transfer of the proteins to customary membranes can be described as at most semiquantitative. A separation of slightly modified proteins (e.g., methyl esters) cannot be effected by means of gel electrophoresis. The use of immunological methods, such as ELISA, is very problematical; as a rule this can only be carried out in aqueous systems. Organic solvents may react with material of microtiter plates and render them unusable. The analytes to be quantified are usually present only in traces. Often, straight hydrophobic proteins are additionally associated with other lipophilic substances (e.g., lipids), which makes quantification according to conventional methods impossible. In ELISA, the components are not separated from one another.
The aim of the invention is to provide a process which allows even strongly hydrophobic proteins, protein fragments and modifications and also strongly hydrophobic peptides to be separated, identified and quantified.
A further aim is to make available a process which makes possible the determination and quantification of, for example, proteins which are slightly modified by acetylation or oxidation.
It is a further aim to provide a process which is suitable, in particular, for the determination of hydrophobic host-cell protein impurities (HCP) in the biotechnological production of hydrophobic proteins, such as the extremely hydrophobic lung surfactant protein SP-C.
These aims are achieved by thin-layer chromatographic separation of a protein mixture dissolved in an organic medium and immunological quantification of the separated proteins.
The object of the invention is therefore a process for the separation, identification and quantification of strongly hydrophobic proteins, protein fragments and modifications and also of strongly hydrophobic peptides, which comprises dissolving the samples to be investigated in an organic medium, subjecting them to thin-layer chromatography and rendering the hydrophobic proteins visible immunologically.
It has surprisingly been found that, for the thin-layer chromatographic separation step, procedures and materials known per se which are adapted to the hydrophobic properties of the proteins to be separated lead to the desired result. Appropriate chromatography plates include all plates having a coating suitable for separation of hydrophobic mixtures in organic media. Examples of such plates are those which have chemically-modified silicon layers, such as HPTLC plates named Diol, CN, NH2, RP-2, RP-8 and RP-18. Of these, those which prove particularly suitable are the HPTLC plates marketed by Merck Darmstadt under the trade name Diol, which have a modified silica matrix. For hydrophobic proteins, suitable mobile phases for thin-layer chromatography are organic solvents and solvent mixtures, e.g., of chloroform and methanol. Mixtures of nonpolar and polar solvents are particularly expedient, possible nonpolar solvents, in particular, being chloroform, methylene chloride and toluene, and polar solvents being shortchain alcohols, particularly methanol, ethanol and isopropanol.
Hydrophobic proteins, protein fragments and modifications and hydrophobic peptides refers to proteins, protein fragments and protein modifications and peptides which, without the aid of a detergent, are poorly soluble in aqueous solvent systems, such as membrane proteins. Surfactant protein C is such a hydrophobic protein. By xe2x80x9cstrongly hydrophobicxe2x80x9d is meant proteins, protein fragments and protein modifications or peptides which are essentially insoluble in an aqueous solvent system.
In the process for preparing surfactant protein C, impurities which consist of host cell proteins are encountered. These have a hydrophobicity similar to that of r-SP-C. Protein fragment refers to a protein which forms part of a complete sequence of a naturally occurring or artificial (synthetic) protein. Protein modification refers to a protein which differs by addition, substitution or deletion of one or more amino acids or chemical modifications, like alkylation, acylation, esterification and oxidation (for example, oxidation of thio radicals) of amino acids, as compared to the naturally occurring or artificial protein. Example: r-SP-C disclosed in WO 95/32992 (copending U.S. application Ser. No. 08/750,194 filed Nov. 27, 1996 now U.S. Pat. No. 5,874,406) is a modification of natural (human) SP-C.
The application of the samples to plates and the separation procedure are carried out in a customary manner, for example by means of commercially available automatic equipment.
Primary antibody/immunological method: The detection step in the process can be carried out in different ways. When an immunological detection method is used, it is carried out with the help of an antibody which (preferably specifically) binds the hydrophobic protein. (HP) to be detected. To this end the thin layer chromatography (TLC) plates are incubated with a (primary) antibody having specificity to the hydrophobic protein. This antibody can carry a label, and this label then provides the basis for detection. If this antibody does not carry a label, a second antibody (carrying a label), having specificity to the first antibody, can be used for detection. Such methods for immunological detection of proteins using antibodies are well known in the art and are, for example, applied in ELISA tests and Western. blotting analyses. The kind of (primary) antibody depends on the kind of hydrophobic protein to be detected. The second antibody will be an anti-xe2x80x9cprimary antibodyxe2x80x9d-antibody.
Besides immunological detection, the detection of the hydrophobic protein on a plate is also achieved by way of conventional staining with a suitable reagent. Reagents used for staining of proteins are well known in the art and include, e.g., Coomassie, Ponceau S and silver.
For preparation for immunological detection, the plates are dried after thin-layer chromatographic separation. For the saturation of nonspecific binding sites, the plates are incubated with a suitable blocking solution, e.g. gelatin or protein. The plates are then incubated with the primary antibody. If this does not carry a label, detection can be carried out with the aid of a labeled second antibody. For detection, all commercially available detection processes can be used. After removal of excess first antibody by washing, incubation is carried out with a labeled second antibody. After washing, the labeled antibodies are detected. They are visualized in a customary manner, e.g., by addition of luminol and hydrogen peroxide, for example with the aid of the ECL (enhanced Chemiluminescence) detection process of Amersham Buchler, which is very sensitive. In a purity analysis, with a view to slight chemical modifications, the substances are also detected on TLC plates directly after separation using customary protein staining reagents.
In the following text, the invention is described by example with the aid of a process for the determination of the host-cell protein impurities in a biotechnological process for the preparation of r-SP-C by means of E. coli. r-SP-C refers to recombinant surfactant protein C or modified derivatives of r-SP-C.
The process is advantageously applied in the detection of hydrophobic impurities (hydrophobic host cell proteins) which are encountered in the production of r-SP-C with the host cells, such as E. coli. Such process for production of r-SP-C is, for example, disclosed in WO 95/32992 (copending application Ser. No. 08/750,194, pages 10 to 12), which shows how to obtain the starting material for the following example. The example (step 1) describes the preparation of antigens (HCP antigens) which are used for the generation of the antibodies (primary antibody) against HCP impurities (step 2). Such antibodies are required in the immunological detection step of the subject process when it is desired to detect HCP impurities.